RNA expression profiling is used to characterize the RNA species present in a sample. Many different techniques are used for this task, each having its own strengths and weaknesses. DNA microarrays are one of the best ‘high-throughput’ techniques for RNA expression profiling. However, most DNA microarrays require extensive sample manipulation. Specifically, sample RNA must be reversed-transcribed into cDNA, then amplified and labeled with one or more fluorophores prior to array hybridization. As a result, biases may be introduced in any of these steps that may artificially skew the results of the microarray analysis. In addition, the current microarray technology is limited in that it is designed to detect only mRNAs.
A major fraction of cellular RNAs, comprise noncoding RNAs, many of which have regulatory functions. Detection of these noncoding RNAs require (or would benefit from) development of novel microarray technology that would not require sample RNA amplification or labeling. Such technology would be desirable, for example, in the detection of microRNAs (miRNAs).
MicroRNAs are small (˜22 nucleotide) regulatory RNAs, that are found in the vast majority of eukaryotic cells. MiRNAs play important roles in plant and animal development, apoptosis, fat metabolism, growth control and hematopoietic differentiation (Lee et al., Cell 116 S89-92, 81 p following S96 (2004); Ruvkun et al., Cell 116, S93-96, 92 p following S96 (2004); Lagos-Quintana et al., Science 294:853-858 (2001); Bartel et al., Cell 116:281-297 (2004); Nelson et al., Trends Biochem. Sci. 28:534-540 (2003); Carrington et al., Science 301:336-338 (2003)). Dysregulation of miRNAs may contribute to human disease including cancer Calin et al., Proc. Natl. Acad. Sci. USA 99:15524-15529 (2002); Michael et al., Mol. Cancer Res. 1:882-891 (2003); Takamizawa et al., Cancer Res. 64:3753-3756 (2004)).
Many individual miRNAs are conserved across widely diverse phyla, indicating their physiological importance. More than 200 miRNAs have been reported thus far from mammals, and miRNAs are estimated to account for ˜1.0% of expressed human genes (Bartel et al., 2004). Most animal miRNAs have the capacity to regulate multiple mRNA targets (Kiriakidou et al., Genes Dev. 18(10); 1165-1178 (2004); reviewed in Bartel et al., 2004). Yet such RNAs cannot be studied using conventional microarray-based techniques.
In mammalian cells, primary miRNA transcripts (pri-miRNAs) are cleaved sequentially in the cell nucleus and transported to the cytoplasm (as pre-miRNAs) where mature miRNAs are generated (Lee et al. Nature 425: 415-419 (2003)). Mature miRNAs guide regulatory proteins to induce translational repression or degradation of specific target mRNAs (Bartel et al., 2004; Murchison et al., Curr. Opin. Cell Biol. 16:223-229 (2004)).
High-throughput miRNA gene expression analysis has proven to be technically challenging. The short length and uniqueness of each miRNA render many conventional tools ineffective. Very small RNAs are difficult to reliably amplify or label without introducing bias (Ohtsuka et al. Eur. J. Biochem. 81:285-291 (1977); Romaniuk et al. Eur. J Biochem. 125:639-643 (1982)). Prior attempts at systematic gene expression analysis have involved dot-blots (Krichevsky et al., RNA 9:1274-1281 (2003)) or Northern blots (e.g., Sempere et al., Genome Biol. 5:R13 (2004); Lim et al., Genes Dev. 17:991-1008 (2003)). Additional assays for sensitive detection of miRNAs or their precursors have been developed, involving realtime quantitative PCR-based analysis of pre-miRNA expression (Schmittgen et al., Nucleic Acids Res. 32:e43 (2004)), or a modification of the Invader assay for miRNA detection and quantitation (Allawi et al., RNA 10:1153- 1161 (2004).
While Northern blots are currently the gold standard of miRNA validation and quantification (Ambros et al., RNA 9:277-279 (2003)), the specificity of the Northern blot technique has received scant critical review. This is surprising considering the widespread use of the method. Short DNA/RNA hybrids demonstrate Tm and binding dynamics that vary significantly with probe and target nucleotide composition, buffer contents, and the time and temperature of hybridization (Dai et al., 2002; Liu et al, 2001; Dorris et al., 2003; Urakawa et al., Appl. Environ. Microbiol. 69:2848-2856 (2003); Guschin et al., Appl. Environ. Microbiol. 63:2397-2402 (1997)). Thus, it is highly probable that “signal intensity” for Northern blots will vary from one miRNA sample to another, as well as from one experiment to another. Moreover, standard Northern blotting does not provide absolute quantification, meaning that each RNA queried must include a standard curve in order to be considered “absolutely quantitative,” and each standard curve must further be run in parallel with each individual experiment (Lim et al., 2003, supra).
Because higher-throughput techniques involving mature miRNAs are needed to further understand the role(s) played by miRNAs in normal and disease tissues, two groups have reported work on microarrays for miRNAs. Croce and colleagues reported an oligonucleotide microarray for miRNA and pre-miRNA profiling (Liu et al., Proc. Natl. Acad. Sci. USA 26:9740-9744 (2004); Liu et al., Proc. Natl. Acad. Sci. USA 32:11755-11760 (2004)), wherein the assay involves the use of a biotinylated primer containing a random octamer sequence at the 3′-end. The Liu et al. primer is used, along with reverse transcriptase, to generate a cDNA library from total RNA. The cDNA is isolated and applied to a microarray containing covalently linked DNA oligonucleotide probes corresponding to 245 human and mouse miRNAs (Liu et al., 2004). Horvitz and colleagues also prepared cDNAs from miRNAs using techniques previously employed for the cloning of miRNAs. This was accomplished by ligating adapters to miRNAs using T4 RNA ligase, followed by R.T-PCR using fluorescently-labeled primers complementary to the adapters (Miska et al., Genome Biol. 5:R68 (2004)). However, while these reported microarray techniques allow for sensitive, specific and high-throughput miRNA expression profiling, e.g., Miska et al., reported a sensitivity of 0.1 fmoles), but the technique also requires PCR amplification of the miRNA sample.
Nevertheless, much remains unknown about miRNA biology. For example, the miRNA genes expressed in most tissues, species, and cell lines, are not known, and the physiological functions-and regulation-of almost all miRNAs remain to be determined. MiRNAs may also play roles in human disease that have not yet been explored. These and other topics will be easier to address experimentally when miRNA gene expression studies become more feasible, and a need in the art has remained until the present invention for simple and reliable DNA microarray techniques that allow for hybridization without RNA amplification or degredation, without the cumbersome steps involved in making and using cDNA, and without the need to label the nucleic acid prior to hybridization.